Our long-term objective is to extend knowledge of microbial aromatic catabolism and its role in the earth's carbon cycle. This information is needed in order to understand the fates of environmental pollutants. Their biodegradability depends upon attach by microbial enzymes normally used for natural products whose chemical structures, like those of pesticides and pollutants, often contain benzene rings. Incomplete degradation, also, may simply result in increased toxicity. Aerobic degradation of aromatics is started by oxygenase attack, and we expect to obtain information about mechanisms of dioxygenases from the kinetics of 2-pyrone-4,6-dicarboxylate formation using gallic acid and its 3-0-Me ether as substrates. We shall purify a flavorprotein monooxygenase that converts anthranilate into 2,3-dihydroxybenzoate, releasing NH3 and incorporating 0 form both 02 and H20. Dr. V. Massey will collaborate with studies of steady-state kinetics. Additional bacterial strains that utilize aromatic hydrocarbons upon receipt of T0L plasmids will be constructed, and their enzymology studied. We used a chemostat to isolate pure cultures that grow with chlorobenzoates, and by similar methods we shall seek strains that utilize other compounds of environmental interest. We shall attempt to increase their range of growth substrates by plasmid transfer. We have proposed a general catabolic sequence for lignin-derived acids, appropriately substituted with 0Me groups, whereby Me0H is furnished to support growth of other strains. This scheme will be further tested using derivatives of cinnamic and phenylacetic acids. Methods will include a new microcalorimetric technique. Catabolic pathways for the versatile eukaryote, T. cutaneum, differ substantially from those for prokaryotes, and these new areas will be explored using alkyl-substituted aromatics. Scientific disciplines involved are: Biochemistry, microbiology of soil and water, organic and analytical chemistry.